Efficient Biotransformations UsingEscherichia coliwithtolCacrABMutations Expressing Cytochrome P450 Genes

Fujii, Tadashi; Fujii, Yoshikazu; Machida, Kazuhiro; Ochiai, Atsushi; Ito, Masashi

doi:10.1271/bbb.80627

Cited by 29 publications

(16 citation statements)

References 19 publications

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“…These data reaffirmed the complexity of the E. coli efflux system: catechin and C3G have very similar molecular backbones, with minor differences in the C-ring and the addition of a glucoside functional group; however, they have very distinct routes of efflux. Also, maintaining a high intracellular pool of substrates by knocking out a substrate-specific efflux pump can significantly enhance biotransformation efficiency (31).…”

Section: Resultsmentioning

confidence: 99%

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Lim

Wong

Bhan

et al. 2015

Appl Environ Microbiol

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bAnthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (؉)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

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Section: Resultsmentioning

confidence: 99%

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Lim

Wong

Bhan

et al. 2015

Appl Environ Microbiol

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“…[24; http://www.schering.de) at a scale of ~100 tons per year. In addition, E. coli -expressed CYP is used to make provastatin from compactin [25], and CYP102 is utilized in the production of epoxyeicosatrienoic acid, leukotoxin B, and eicosanoid epoxides [26]. Many synthetic steps of antibiotics involve bacterial P450s, e.g.…”

Section: Applications Of Cytochrome P450 Biocatalystsmentioning

confidence: 99%

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Kumar

2010

Expert Opinion on Drug Metabolism & Toxicology

149

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Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts.

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“…However, fusion constructs between P450sca-2 and the P450BM3 reductase or the reductase module of the Rhodococcus RhF P450-reductase fusion enzyme also did not result in compactin to pravastatin conversion. Recently, P450sca-2 was successfully mutated and expressed in E. coli (36,37). Interestingly, the authors did not select a fusion to the Rhf reductase as the reductase crystal structure is unknown (38), but selected the Pseudomonas putida reductase/putidaredoxin system (38, 39), leading to 377.5 mg/L pravastatin from this three-component redox system.…”

Section: Discussionmentioning

confidence: 99%

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

McLean

Hans

Meijrink

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

121

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The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6-epi-pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450 Prava bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450 Prava fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.pravastatin | P450 engineering | fermentation | statin | cholesterol

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Efficient Biotransformations UsingEscherichia coliwithtolCacrABMutations Expressing Cytochrome P450 Genes

Cited by 29 publications

References 19 publications

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

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